Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer|转dbr2基因提高青蒿中青蒿素含量的方法
Entry posted by Roger in Artemisinin Patent｜青蒿素专利
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The invention provides a method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer in the field of biotechnology. The method includes the steps: cloning genes of artemisinic aldehyde delta 11(13) DBR2 from the sweet wormwood, constructing a plant expression vector containing the DBR2 genes, using agrobacterium tumefaciens for mediation to transfer the DBR2 genes into the sweet wormwood so that plants regenerate, using PCR (polymerase chain reaction) to detect integration conditions of the exogenous targeted DBR2 genes, determining the artemisinin content in the transgenic sweet wormwood by means of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and screening the transgenic sweet wormwood plants with the improved artemisinin content. The artemisinin content in the obtained transgenic sweet wormwood can be remarkably increased and is 2.83 times maximally of that of a non-transgenic control plant, and accordingly the method for increasing the artemisinin content in the sweet wormwood lays a foundation for large-scale artemisinin production by the aid of the transgenic sweet wormwood.
本发明是一种生物技术领域的转DBR2基因提高青蒿中青蒿素含量的方法。本发明从青蒿中克隆青蒿醛Δ11(13)还原酶DBR2的基因，构建含DBR2基因的植物表达载体，用根癌农杆菌介导，将DBR2基因转入青蒿并再生出植株，PCR检测外源目的基因DBR2的整合情况，高效液相色谱法及蒸发光散射检测器测定(HPLC-ELSD)转基因青蒿中青蒿素的含量，筛选获得青蒿素含量提高的转基因青蒿植株。本发明获得的转基因青蒿中青蒿素的含量显著提高，最高达到非转化对照植株的2.83倍，从而提供了一种提高青蒿中青蒿素含量的方法，为利用转基因青蒿大规模生产青蒿素打下了基础。
Description
Turn the method that DBR2 gene improves content of artemisinin in sweet wormwood
Technical field
What the present invention relates to is a kind of method of raising artemislnin content of biological technical field, particularly a kind ofly turns the method that DBR2 gene improves content of artemisinin in sweet wormwood.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin (artemisinin) is a kind of sesquiterpene lactones compound containing peroxide bridge structure be separated from its over-ground part, be the medicine of the most effectively treating malaria of generally acknowledging in the world at present, particularly for encephalic malaria and anti-chloroquine malaria, there is quick-acting and feature that is low toxicity.At present, the method for the most effectively treating malaria of world health organisation recommendations is exactly Artemisinin conjoint therapy (ACTs).In addition, along with progressively going deep into Artemisinin pharmacological research, scientist finds that Artemisinin and derivative thereof also have anti-inflammatory, schistosomicide, antitumor and immunoregulatory function.Visible Artemisinin is a kind of natural drug of great potential.
The main source of current Artemisinin extracts from the over-ground part of sweet wormwood plant, but the content of Artemisinin in Artemisia annuna is very low, in different planting environment and varieties of plant, its average content is at the 0.01-1% of sweet wormwood leaf dry weight, the large-scale commercial of this medicine is produced and is restricted.Due to Artemisinin complex structure, synthetic difficulty is large, and yield poorly, cost is high, does not have feasibility.The method of someone trial tissue culture and cell engineering produces Artemisinin, however Artemisinin in callus, content is lower than 0.1% of dry weight, the highest in bud also only have 0.16% of dry weight, and great majority research does not detect Artemisinin in root.Therefore utilize tissue culture and cell engineering not high to the feasibility of producing Artemisinin yet.
Through finding prior art literature search, Waleerat Banyai etc. are in " Plant Cell Tissue and OrganCulture " (plant cell tissue's organ culture), within 2010,103 volume 255-265 pages have delivered the paper being entitled as " Overexpressionof farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth ofArtemisia annua L. " (" overexpression farnesyl pyrophosphate synthase gene can affect the content of Artemisinin in Artemisia annuna and the growth of sweet wormwood "), report by crossing most representation farnesyl pyrophosphate synthase (farnesyl pyrophosphatesynthase, FPS), the content of render transgenic Artemisinin in Artemisia annuna improves 2.5-3.6 doubly, but still only have about 1.3%.But, plant genetic engineering is improve the content of Artemisinin in Artemisia annuna to provide a feasible method.
Sweet wormwood aldehyde Δ 11 (13) reductase enzyme (artemisinic aldehyde Δ 11 (13) double bondreductase in prior art, DBR2) being a key enzyme in artemisinin synthesis approach, is the important target spot of Artemisinin metabolic engineering.Adopt genetic engineering means, transform sweet wormwood with key gene DBR2, the biosynthetic speed limit bottleneck of Artemisinin will be broken, obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a new way.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, provide a kind of and turn the method that DBR2 gene improves content of artemisinin in sweet wormwood.The key gene clone that the present invention relates to, vector construction, genetic transformation, Molecular Detection, Artemisinin extraction and assay are for the present invention, establishing the stable method improving content of artemisinin in sweet wormwood, establishing solid basis for utilizing sweet wormwood scale operation Artemisinin.
The present invention is achieved by the following technical solutions:
Turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, comprise following concrete steps:
(1) gene clone method is adopted to obtain sweet wormwood key gene DBR2;
(2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene;
(3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained;
(4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR;
(5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves.
Preferably, in described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ ID NO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification.
Preferably, in described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification.
Preferred further, described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme.
Preferably, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant.
Preferred further, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, blots surface-moisture with aseptic thieving paper, is inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
Preferred further, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast.
Preferred further, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained.
Preferably, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant.
Preferably, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, moving phase select volume ratio be 70: 30 methyl alcohol: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar.
The method turning DBR2 gene raising content of artemisinin in sweet wormwood of the present invention; adopt gene engineering method; key gene DBR2 is imported in sweet wormwood plant; obtain the transgene abrotanum strain that artemislnin content significantly improves; the content turning DBR2 gene Artemisinin in Artemisia annuna can reach the 22.6mg/g of dry weight; be 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight), this invention provides high yield for the large-scale production for Artemisinin, to stablize source new drugs significant.
Accompanying drawing explanation
Fig. 1 is the content detection result figure of Artemisinin in sweet wormwood plant of the present invention.
Embodiment
Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
embodiment
Step one, the clone of sweet wormwood DBR2 gene
(1) extraction of sweet wormwood genome total serum IgE
Get 100-200mg sweet wormwood young leaflet tablet, after liquid nitrogen flash freezer, grind with mortar rapidly, add and fill 1mL TRlzol (TRlzol Reagents, GIBCOBRL, USA) 1.5mL Eppendorf pipe in, fully after vibration, 5min is put in ambient temperatare, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature places 2-3min, 12,000rmp centrifugal 15min at 4 DEG C; Supernatant liquor (about 600 μ L) is sucked in clean 1.5mL Eppendorf pipe, adds isopyknic Virahol, put upside down mixing, after ambient temperatare puts 10min, 12,000rmp centrifugal 10min at 4 DEG C; Abandon supernatant, add 1mL 75% ethanol purge, after vibration, 7,500rmp centrifugal 5min at 4 DEG C; Be dissolved in appropriate (30-40 μ L) RNAase-free water after drying at room temperature 10-15min; By denaturing formaldehyde gel electrophoresis qualification total serum IgE quality, then on spectrophotometer, measure rna content.
(2) clone of sweet wormwood DBR2 gene
Obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, according to the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of described sweet wormwood DBR2 gene, design amplifies upstream primer (DNA sequence dna shown in SEQ ID NO.2) and the downstream primer (DNA sequence dna shown in SEQ ID NO.3) of complete encoder block, and on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the first described chain cDNA for template, check order after pcr amplification.Determined dna sequence has been checked order by Shanghai Ying Jun biotechnology Services Co., Ltd.Sequencing result shows, the sequence of cloning is consistent with the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of the sweet wormwood DBR2 gene reported in GenBank.
The present embodiment adopts gene clone method from sweet wormwood, obtain the correct Artemisinin biosynthesizing key gene DBR2 of sequence, for providing an important key gene by turning DBR2 gene raising content of artemisinin in sweet wormwood.
Step 2, containing the structure of the plant binary expression vector of DBR2 gene
(1) structure of intermediate carrier pMDT18-DBR2
Select pMDT18 carrier (Takara, Dalian) for primary element, build intermediate carrier pMDT18-DBR2.Particularly, by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme, being checked order by Shanghai Ying Jun biotechnology Services Co., Ltd confirms the exactness of gene.
(2) containing the structure of the plant expression vector of DBR2 gene
For expression vector, above-mentioned DBR2 gene is connected into the restriction enzyme site position of its correspondence with described FSN (FSN expression vector obtains for transforming on pCAMBIA2300 expression vector and preserves).Particularly, XmaI and SacI double digestion intermediate carrier pMDT18-dbr2 and expression vector FSN.Reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification.
Artemisinin biosynthetic pathway key gene DBR2 is connected to expression regulation sequence by the present embodiment operably, and form the plant expression vector containing DBR2 gene, this expression vector can be used for the content being improved Artemisinin in Artemisia annuna by metabolic engineering strategies.
Step 3, containing the acquisition of DBR2 gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
The above-mentioned plant binary expression vector containing DBR2 gene is proceeded to agrobacterium tumefaciens (as EHA105, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar 1), and performing PCR of going forward side by side is verified.
Step 4, Agrobacterium tumefaciens mediated DBR2 gene transformation sweet wormwood
(1) preculture of explant
Seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in MS (the Murashige and Skoog without hormone, 1962), in solid medium, 25 DEG C, 16h/8h (light/dark) illumination cultivation, can obtain sweet wormwood aseptic seedling.Grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
(2) Dual culture of Agrobacterium and explant
By described leaf explant, forward in Dual culture substratum (1/2MS+AS 100 μm of ol/L), drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted, 28 DEG C of light culture 3 days with bacterium liquid.With drip the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene leaf explant for contrast.
(3) screening of resistance regeneration plant
The described Dual culture sweet wormwood explant of 3 days is transferred in germination screening culture medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h (light/dark) illumination cultivation, every two weeks succeeding transfer culture once, can obtain Kan resistance Multiple Buds after 2-3 subculture.Well-grown resistance Multiple Buds is cut proceed to root media (1/2MS+Cb 125mg/L) is cultured to and take root, thus obtain Kan resistance regeneration sweet wormwood plant.
Step 5, the PCR of transgene abrotanum plant detects
Forward primer is designed respectively and reverse primer detects goal gene according to goal gene place expression cassette p35s-dbr2-nos sequence p35s and dbr2.Result shows, the PCR special primer designed by utilization, can amplify the specific DNA fragment of 458bp.And with non-transformed sweet wormwood genomic dna for template time, do not amplify any fragment.
The present embodiment is by described plant expression vector transform Agrobacterium tumefaciens, obtain the Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector for transforming sweet wormwood, Agrobacterium tumefaciens strain constructed by utilization transforms sweet wormwood, obtains the transgene abrotanum plant detected through PCR.The acquisition of transgene abrotanum plant is screen the sweet wormwood strain obtaining higher artemislnin content to provide direct material.
Step 6, utilizes HPLC-ELSD to measure artemislnin content in transgene abrotanum
(1) preparation of HPLC-ELSD condition and system suitability and standardized solution
HPLC: adopt water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel post (Symmetry ShieldTM C18,5 μm, 250 × 4.6mm, Waters), moving phase is methyl alcohol: water, methyl alcohol: the volume ratio of water is 70: 30, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number calculates by Artemisinin peak and is not less than 2000.
ELSD: adopt water alliance 2420 system, light scattering detector drift tube temperature 40 DEG C, scale-up factor (gain) is 7, nebulizer gas pressure 5bar;
Precision takes Artemisinin standard substance (Sigma company) 2.0mg 1mL methyl alcohol and dissolves completely, obtains 2mg/mL Artemisinin standard solution, be stored in-20 DEG C for subsequent use.
In the present embodiment, moving phase is methyl alcohol: water, and when ratio is 70%: 30%, the retention time of Artemisinin is 5.1min, and peak type is good.Theoretical plate number calculates by Artemisinin and is not less than 2000.
(2) making of typical curve
By described reference substance solution difference sample introduction 2 μ L under corresponding chromatographic condition, 4 μ L, 6 μ L, 8 μ L, 10 μ L record collection of illustrative plates and chromatographic parameters, carry out regression analysis respectively with peak area (Y) to standard substance content (X, μ g).By research, in the present embodiment, Artemisinin presents good log-log linear relationship within the scope of 4-20 μ g.The log-log equation of linear regression of Qinghaosu is Y=5.404e+0000X+1.858e+0000, R 2=0.999184.
(3) preparation of sample and the mensuration of artemislnin content
The leaching process of Artemisinin is based on the method reported in Van Nieuwerburgh et al. (2006): the sweet wormwood blade (1-2g fresh weight) taking a morsel fresh, be immersed in 50ml test tube in 10ml chloroform and swayed 1 minute, leach liquor is poured in new test tube and make chloroform volatilize completely, get 3ml dehydrated alcohol and fully dissolve extract, filter after filter filters through 0.22 μm and detect for HPLC.Meanwhile, 60 DEG C of baking ovens are put in the blade collection after chloroform extraction dries, weigh (dry weight calculating sweet wormwood blade);
HPLC-ELSD is adopted to measure artemislnin content, sample feeding volume is 20 μ L, the artemislnin content (mg) in sample is gone out according to peak area substitution linear regression Equation for Calculating, again divided by artemisia leaf dry weight (g) of sample, thus calculate the content of Artemisinin in sweet wormwood plant.
Turn DBR2 gene in an embodiment and significantly improve content of artemisinin in sweet wormwood.The content turning DBR2 gene blue or green senior middle school Artemisinin can reach the 22.6mg/g (as shown in Figure 1) of dry weight, is 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight).
The present embodiment adopts HPLC-ELSD method to determine artemislnin content in transgene abrotanum, adopts the metabolic engineering strategies transforming DBR2 gene to obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a kind of Perfected process.
Claims (7)
1. turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, it is characterized in that, comprise following concrete steps:
(1) gene clone method is adopted to obtain sweet wormwood key gene DBR2;
(2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene;
(3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained;
(4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR;
(5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves;
In described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ IDNO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification,
In described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extracts plasmid and does PCR detection and digestion verification; Described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme.
2. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant.
3. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, then soaks 20min with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming.
4. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast.
5. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained.
6. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, is characterized in that, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant.
7. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, and moving phase selects volume ratio to be the methyl alcohol of 70:30: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar.
转DBR2基因提高青蒿中青蒿素含量的方法
技术领域
本发明涉及的是一种生物技术领域的提高青蒿素含量的方法，特别是一种转DBR2基因提高青蒿中青蒿素含量的方法。
背景技术
青蒿(Artemisia annua L.)是菊科蒿属的一年生草本植物。青蒿素(artemisinin)是从其地上部分分离的一种含有过氧桥结构的倍半萜内酯化合物，是目前世界上公认的最有效的治疗疟疾的药物，特别是对于脑型疟疾和抗氯喹疟疾具有速效和低毒的特点。目前，世界卫生组织推荐的最有效的治疗疟疾的方法就是青蒿素联合疗法(ACTs)。另外，随着对青蒿素药理研究的逐步深入，科学家发现青蒿素及其衍生物还具有抗炎、抗血吸虫、抗肿瘤以及免疫调节的功能。可见青蒿素是一种极具潜力的天然药物。
目前青蒿素的主要来源是从青蒿植株的地上部分提取，然而青蒿中青蒿素的含量非常低，不同种植环境和种植品种中其平均含量在青蒿叶片干重的0.01-1％，使得这种药物的大规模商业化生产受到了限制。由于青蒿素结构复杂，人工合成难度大，产量低，成本高，不具有可行性。有人尝试用组织培养和细胞工程的方法来生产青蒿素，然而青蒿素在愈伤组织中含量低于干重的0.1％，在芽中最高也只有干重的0.16％，而大多数研究在根中没有检测到青蒿素。因此利用组织培养及细胞工程来生产青蒿素的可行性也不高。
经对现有技术文献检索发现，Waleerat Banyai等在《Plant Cell Tissue and OrganCulture》(植物细胞组织器官培养)，2010年103卷255-265页发表了题为“Overexpressionof farnesyl pyrophosphate synthase(FPS)gene affected artemisinin content and growth ofArtemisia annua L.”(“过量表达法尼基焦磷酸合酶基因能影响青蒿中青蒿素的含量及青蒿的生长”)的论文，报道通过过最表达法尼基焦磷酸合酶(farnesyl pyrophosphatesynthase，FPS)，使转基因青蒿中青蒿素的含量提高了2.5-3.6倍，但仍然只有1.3％左右。不过，植物基因工程为提高青蒿中青蒿素的含量提供了一条可行的方法。
现有技术中青蒿醛Δ11(13)还原酶(artemisinic aldehydeΔ11(13)double bondreductase，DBR2)是青蒿素合成途径中的一个关键酶，是青蒿素代谢工程的重要靶点。采用基因工程手段，用关键酶基因DBR2转化青蒿，将打破青蒿素生物合成的限速瓶颈，获得青蒿素高产的青蒿植株，为规模化生产青蒿素提供一条新途径。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种转DBR2基因提高青蒿中青蒿素含量的方法。本发明涉及的关键酶基因克隆、载体构建、遗传转化、分子检测、青蒿素提取及含量测定用于本发明，建立了稳定提高青蒿中青蒿素含量的方法，为利用青蒿大规模生产青蒿素奠定坚实的基础。
本发明是通过以下技术方案实现的：
一种转DBR2基因提高青蒿中青蒿素含量的方法，包括如下具体步骤：
(1)采用基因克隆方法获得青蒿关键酶基因DBR2；
(2)把所述DBR2基因连结于表达调控序列，构建含DBR2基因的植物表达载体；
(3)将所述含DBR2基因的植物表达载体转化根癌农杆菌，获得含所述DBR2基因植物表达载体的根癌农杆菌菌株；
(4)利用所述含DBR2基因植物表达载体的根癌农杆菌菌株转化青蒿，获得经PCR检测的整合外源目的基因DBR2的转基因青蒿植株；
(5)对所述转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株。
优选的，在所述步骤(1)中，所述基因克隆方法包括以下步骤：提取青蒿基因组总RNA，将所获的青蒿基因组总RNA通过反转录酶XL反转录获得第一链cDNA，根据SEQ ID NO.1所示的DNA序列，设计扩增出完整编码框的上游引物和下游引物，所述上游引物为SEQ ID NO.2所示的DNA序列，所述下游引物为SEQ ID NO.3所示的DNA序列，并在所述上游和下游引物上分别引入限制性内切酶位点以便构建表达载体，以所述的第一链cDNA为模板，经PCR扩增后进行测序。
优选的，在所述步骤(2)中，所述构建含DBR2基因的植物表达载体包括以下步骤：先构建中间载体pMDT18-DBR2，再用XmaI和SacI酶切中间载体pMDT18-DBR2和表达载体FSN，回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证。
进一步优选的，所述构建中间载体pMDT18-DBR2包括以下具体步骤：通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上。
优选的，在所述步骤(4)中，所述转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。
进一步优选的，所述预培养包括以下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃光照培养，即可获得青蒿无菌苗，待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。
进一步优选的，所述共培养包括以下步骤：将所述叶片外植体转到共培养培养基中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天，以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。
进一步优选的，所述筛选包括以下步骤：将所述共培养3天的青蒿外植体转入到发芽筛选培养基上于25℃光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将所述生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，即可获得Kan抗性再生青蒿植株。
优选的，在所述步骤(4)中，所述PCR检测包括以下步骤：设计合成DBR2基因的引物；进行DNA扩增；紫外线下观察，若目的条带为阳性，则该株系即为所述转基因青蒿植株。
优选的，在所述步骤(5)中，所述HPLC-ELSD测定包括以下条件：所用色谱柱为C-18反相硅胶柱，流动相选用体积比为70∶30的甲醇∶水，柱温30℃，流速1.0mL/min，进样量20μL，蒸发光散射检测器漂移管温度40℃，放大系数为7，载气压力5bar。
本发明的转DBR2基因提高青蒿中青蒿素含量的方法，采用基因工程方法，将关键酶基因DBR2导入青蒿植株中，获得了青蒿素含量显著提高的转基因青蒿株系，转DBR2基因青蒿中青蒿素的含量最高可达到干重的22.6mg/g，是非转化普通青蒿(8mg/g干重)的2.83倍，该发明对于为青蒿素的规模化生产提供高产、稳定新药源具有重要意义。
附图说明
图1为本发明的青蒿植株中青蒿素的含量检测结果图。
具体实施方式
下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例
步骤一，青蒿DBR2基因的克隆
(1)青蒿基因组总RNA的提取
取100-200mg青蒿幼嫩叶片，用液氮速冻后，迅速用研钵研碎，加入盛有1mL TRlzol(TRlzol Reagents，GIBCOBRL，USA)的1.5mL Eppendorf管中，充分振荡后，于室温下放置5min，加200μL氯仿，用力振荡15sec，室温放置2-3min后，于4℃下12,000rmp离心15min；将上清液(约600μL)吸入干净的1.5mL Eppendorf管中，加入等体积的异丙醇，颠倒混匀，室温下放置10min后，于4℃下12,000rmp离心10min；弃上清，加1mL 75％乙醇清洗，振荡后，于4℃下7,500rmp离心5min；室温干燥10-15min后溶于适量(30-40μL)RNAase-free水中；用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。
(2)青蒿DBR2基因的克隆
将所获的青蒿基因组总RNA通过反转录酶XL(AMV)反转录获得第一链cDNA，根据所述青蒿DBR2基因的编码序列(SEQ ID NO.1所示的DNA序列)，设计扩增出完整编码框的上游引物(SEQ ID NO.2所示的DNA序列)和下游引物(SEQ ID NO.3所示的DNA序列)，并在上游和下游引物上分别引入限制性内切酶位点(这可视选用的载体而定)，以便构建表达载体。以所述的第一链cDNA为模板，经PCR扩增后进行测序。DNA序列测定由上海英骏生物技术服务有限公司测序完成。测序结果表明，所克隆的序列与GenBank中所报道的青蒿DBR2基因的编码序列(SEQ ID NO.1所示的DNA序列)一致。
本实施例采用基因克隆方法从青蒿中获得序列正确的青蒿素生物合成关键酶基因DBR2，为通过转DBR2基因提高青蒿中青蒿素含量提供了一个重要关键酶基因。
步骤二，含DBR2基因的植物双元表达载体的构建
(1)中间载体pMDT18-DBR2的构建
选用pMDT18载体(Takara，Dalian)为基本元件，构建中间载体pMDT18-DBR2。具体地，通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上，由上海英骏生物技术服务有限公司测序确认基因的正确性。
(2)含DBR2基因的植物表达载体的构建
以所述的FSN(FSN表达载体为在pCAMBIA2300表达载体上改造获得并保存)为表达载体，将上述DBR2基因连入其对应的酶切位点位置。具体地，XmaI和SacI双酶切中间载体pMDT18-dbr2和表达载体FSN。回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证。
本实施例将青蒿素生物合成途径关键酶基因DBR2可操作性地连接于表达调控序列，形成含DBR2基因的植物表达载体，该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素的含量。
步骤三，含DBR2基因双元植物表达载体根癌农杆菌工程菌的获得
将上述含DBR2基因的植物双元表达载体转入根癌农杆菌(如EHA105，为市场有公开出售的生物材料，可以从澳大利亚CAMBIA公司购得，菌株编号为Gambar 1)，并进行PCR验证。
步骤四，根癌农杆菌介导DBR2基因转化青蒿
(1)外植体的预培养
青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS(Murashige and Skoog，1962)固体培养基中，25℃、16h/8h(light/dark)光照培养，即可获得青蒿无菌苗。待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。
(2)农杆菌与外植体的共培养
将所述的叶片外植体，转到共培养培养基(1/2MS+AS 100μmol/L)中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天。以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。
(3)抗性再生植株的筛选
将所述的共培养3天的青蒿外植体转入到发芽筛选培养基(MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L)上于25℃、16h/8h(light/dark)光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根，从而获得Kan抗性再生青蒿植株。
步骤五，转基因青蒿植株的PCR检测
根据目的基因所在表达盒p35s-dbr2-nos序列p35s和dbr2分别设计正向引物和反向引物对目的基因进行检测。结果表明，利用所设计的PCR特异引物，能扩增出458bp的特异DNA片段。而以非转化青蒿基因组DNA为模板时，没有扩增出任何片段。
本实施例将所述的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含DBR2基因植物表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测的转基因青蒿植株。转基因青蒿植株的获得为筛选获得较高青蒿素含量的青蒿株系提供了直接素材。
步骤六，利用HPLC-ELSD测定转基因青蒿中青蒿素含量
(1)HPLC-ELSD条件及系统适用性以及标准溶液的配制
HPLC：采用water alliance 2695系统，色谱柱为C-18反相硅胶柱(Symmetry ShieldTM C18，5μm，250×4.6mm，Waters)，流动相为甲醇∶水，甲醇∶水的体积比为70∶30，柱温30℃，流速1.0mL/min，进样量10μL，灵敏度(AUFS＝1.0)，理论塔板数按青蒿素峰计算不低于2000。
ELSD：采用water alliance 2420系统，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar；
精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解，得到2mg/mL青蒿素标准品溶液，保存于-20℃备用。
本实施例中流动相为甲醇∶水，比例为70％∶30％时，青蒿素的保留时间为5.1min，峰型良好。理论塔板数按青蒿素计算不低于2000。
(2)标准曲线的制作
将所述对照品溶液在相应色谱条件下分别进样2μL，4μL，6μL，8μL，10μL记录图谱及色谱参数，分别以峰面积(Y)对标准品含量(X，μg)进行回归分析。通过研究，本实施例中青蒿素在4-20μg范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为Y＝5.404e+0000X+1.858e+0000，R2＝0.999184。
(3)样品的制备和青蒿素含量的测定
青蒿素的提取过程基于Van Nieuwerburgh et al.(2006)中报道的方法：取少量新鲜的青蒿叶片(1-2g鲜重)，于50ml试管中将其浸没在10ml氯仿中摇荡1分钟，将浸出液倒入新的试管中使氯仿挥发完全，取3ml无水乙醇充分溶解提取物，经0.22μm过滤滤头过滤后用于HPLC检测。同时，氯仿提取后的叶片收集放入60℃烘箱进行烘干，称重(计算青蒿叶片的干重)；
采用HPLC-ELSD测定青蒿素含量，样品进样体积为20μL，根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg)，再除以样品的青蒿叶干重(g)，从而计算出青蒿植株中青蒿素的含量。
在实施例中转DBR2基因显著提高青蒿中青蒿素含量。转DBR2基因青高中青蒿素的含量最高可达到干重的22.6mg/g(如图1所示)，是非转化普通青蒿(8mg/g干重)的2.83倍。
本实施例采用HPLC-ELSD法测定了转基因青蒿中青蒿素含量，采用转化DBR2基因的代谢工程策略获得了青蒿素高产的青蒿植株，为规模化生产青蒿素提供了一种理想方法。
Claims (7)
1.一种转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，包括如下具体步骤：
(1)采用基因克隆方法获得青蒿关键酶基因DBR2；
(2)把所述DBR2基因连结于表达调控序列，构建含DBR2基因的植物表达载体；
(3)将所述含DBR2基因的植物表达载体转化根癌农杆菌，获得含所述DBR2基因植物表达载体的根癌农杆菌菌株；
(4)利用所述含DBR2基因植物表达载体的根癌农杆菌菌株转化青蒿，获得经PCR检测的整合外源目的基因DBR2的转基因青蒿植株；
(5)对所述转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株；
在所述步骤(1)中，所述基因克隆方法包括以下步骤：提取青蒿基因组总RNA，将所获的青蒿基因组总RNA通过反转录酶XL反转录获得第一链cDNA，根据SEQ IDNO.1所示的DNA序列，设计扩增出完整编码框的上游引物和下游引物，所述上游引物为SEQ ID NO.2所示的DNA序列，所述下游引物为SEQ ID NO.3所示的DNA序列，并在所述上游和下游引物上分别引入限制性内切酶位点以便构建表达载体，以所述的第一链cDNA为模板，经PCR扩增后进行测序；
在所述步骤(2)中，所述构建含DBR2基因的植物表达载体包括以下步骤：先构建中间载体pMDT18-DBR2，再用XmaI和SacI酶切中间载体pMDT18-DBR2和表达载体FSN，回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证；所述构建中间载体pMDT18-DBR2包括以下具体步骤：通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上。
2.根据权利要求1所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，在所述步骤(4)中，所述转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。
3.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述预培养包括以下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃光照培养，即可获得青蒿无菌苗，待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。
4.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述共培养包括以下步骤：将所述叶片外植体转到共培养培养基中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天，以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。
5.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述筛选包括以下步骤：将所述共培养3天的青蒿外植体转入到发芽筛选培养基上于25℃光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将所述生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，即可获得Kan抗性再生青蒿植株。
6.根据权利要求1所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，在所述步骤(4)中，所述PCR检测包括以下步骤：设计合成DBR2基因的引物；进行DNA扩增；紫外线下观察，若目的条带为阳性，则该株系即为所述转基因青蒿植株。
7.根据权利要求1所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，在所述步骤(5)中，所述HPLC-ELSD测定包括以下条件：所用色谱柱为C-18反相硅胶柱，流动相选用体积比为70:30的甲醇:水，柱温30℃，流速1.0mL/min，进样量20μL，蒸发光散射检测器漂移管温度40℃，放大系数为7，载气压力5bar。
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